Sources of Ca²⁺ in relation to generation of acetylcholine-induced endothelium-dependent hyperpolarization in rat mesenteric artery

Abstract

1. The aim of the present study was to identify the sources of Ca²⁺ contributing to acetylcholine (ACh)-induced release of endothelium-derived hyperpolarizing factor (EDHF) from endothelial cells of rat mesenteric artery and to assess the pathway involved. The changes in membrane potentials of smooth muscles by ACh measured with the microelectrode technique were evaluated as a marker for EDHF release.

2. ACh elicited membrane hyperpolarization of smooth muscle cells in an endothelium-dependent manner. The hyperpolarizing response was not affected by treatment with 10 μM indomethacin, 300 μM N^G-nitro-L-arginine or 10 μM oxyhaemoglobin, thereby indicating that the hyperpolarization is not mediated by prostanoids or nitric oxide but is presumably by EDHF.

3. In the presence of extracellular Ca²⁺, 1 μM ACh generated a hyperpolarization composed of the transient and sustained components. By contrast, in Ca²⁺-free medium, ACh produced only transient hyperpolarization.

4. Pretreatment with 100 nM thapsigargin and 3 μM cyclopiazonic acid, endoplasmic reticulum Ca²⁺-ATPase inhibitors, completely abolished ACh-induced hyperpolarization. Pretreatment with 20 mM caffeine also markedly attenuated ACh-induced hyperpolarization. However, the overall pattern and peak amplitude of hyperpolarization were unaffected by pretreatment with 1 μM ryanodine.

5. In the presence of 5 mM Ni²⁺ or 3 mM Mn²⁺, the hyperpolarizing response to ACh was transient, and the sustained component of hyperpolarization was not observed. On the other hand, 1 μM nifedipine had no effect on ACh-induced hyperpolarization.

6. ACh-induced hyperpolarization was nearly completely eliminated by 500 nM U-73122 or 200 μM 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate, inhibitors of phospholipase C, but was unchanged by 500 nM U-73343, an inactive form of U-73122. Pretreatment with 20 nM staurosporine, an inhibitor of protein kinase C, did not modify ACh-induced hyperpolarization.

7. These results indicate that the ACh-induced release of EDHF from endothelial cells of rat mesenteric artery is possibly initiated by Ca²⁺ release from inositol 1,4,5-trisphosphate (IP₃)-sensitive Ca²⁺ pool as a consequence of stimulation of phospholipid hydrolysis due to phospholipase C activation, and maintained by Ca²⁺ influx via a Ni²⁺- and Mn²⁺-sensitive pathway distinct from L-type Ca²⁺ channels. The Ca²⁺-influx mechanism seems to be activated following IP₃-induced depletion of the pool.

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